The experiments described in this application are designed to provide a clear understanding of the chemical and physical parameters that regulate electron-transfer (ET) reactions in proteins. ET reactions are known to key steps in many biological processes. Biological electron transfers are characterized by several unique features not found in small-molecule reactions: the reactions proceed at low driving forces (less than 200 mV), but with high efficiencies; the redox sites tend to be metal centers buried inside polypeptide matrices, though often with exposed metal-ligand edges; because of this peptide sheath, the redox centers are generally separated by long distances (10-25 angstroms); and the specificity of binding sites restricts the mutual orientations of the proteins, as well as the redox sites. The challenge, then, is to comprehend how the physical and chemical properties of biomolecules produce highly efficient electron-transport systems. During the next five years, systematic investigations of intramolecular ET reactions will be performed with three proteins: Pseudomonas aeruginosa azurin, human myoglobin, and bovine cytochrome b5. In all three cases, site directed mutagenesis will be used to prepare proteins specifically designed to characterize the role of nuclear reorientation and to reveal the mechanism of donor-acceptor electronic coupling in long range protein ET reactions. A new technique for the measurement of intramolecular ET rates will be developed. Luminescent metal complexes bound to surface residues of metalloproteins will be excited with laser pulses and quenched with irreversible ET reagents. The protein-based quenching product will relax via intramolecular ET. Chemical modifications of the surface-bound metal complexes will vary the driving force for the intramolecular ET reaction. The driving-force and temperature dependences of ET rates in these modified proteins will reveal nuclear reorganization energies and electronic coupling strengths for the reactions. Two models for electronic have evolved: one predicts that the coupling strength decays exponentially with direct donor-acceptor distance; and other suggests that electronic coupling is mediated along specific pathways (comprised of covalent and noncovalent interactions) between donor and acceptor. In order to resolve this issue, site directed mutagenesis will be used to prepare mutant proteins that can be modified by attachment of metal complexes to selected surface residues. Modified proteins with similar direct donor-acceptor separations, but significantly different coupling pathways can be identified. The Q12H and K122H mutants of azurin illustrate the point. The exponential-decay model predicts nearly identical ET rates for the two derivatives; the pathway model predicts that the rate for the Q21H derivative will be slower then that if the K122H derivative by a factor of 10 to the fourth power. Comparisons of this type will clarify the mechanism of electronic coupling in long-range ET reactions in proteins.